0090-9556/04/3209-1040–1047$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics
DMD 32:1040–1047, 2004
Vol. 32, No. 9
1417/1169877
Printed in U.S.A.
CARRIER-MEDIATED UPTAKE OF H2-RECEPTOR ANTAGONISTS BY THE RAT CHOROID
PLEXUS: INVOLVEMENT OF RAT ORGANIC ANION TRANSPORTER 3
Yoshinori Nagata, Hiroyuki Kusuhara, Shuichi Hirono, Hitoshi Endou, and Yuichi Sugiyama
Graduate School of Pharmaceutical Sciences, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan (Y.N., H.K., Y.S.);
School of Pharmaceutical Sciences, Kitasato University, Shirokane, Mitano-ku, Tokyo, Japan (S.H.); and Department of
Pharmacology and Toxicology, Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo, Japan (H.E.)
Received February 17, 2004; accepted May 20, 2004
This article is available online at http://dmd.aspetjournals.org
ABSTRACT:
famotidine remained at the highest concentration examined (1 mM).
The Ki value of ranitidine for the uptake of cimetidine by the isolated
CP (50 ␮M) was similar to its own Km value, suggesting that they
share the same transporter for their uptake. The inhibition potency of
organic anions such as benzylpenicillin, estradiol 17␤-glucuronide,
p-aminohippurate, and estrone sulfate for the uptake of cimetidine by
the isolated rat CP was similar to that for benzylpenicillin, the uptake
of which has been hypothesized to be mediated by rOat3, whereas a
minimal effect by tetraethylammonium excludes involvement of organic cation transporter(s). These results suggest that rOat3 is the
most likely candidate transporter involved in regulating the CSF concentration of H2-receptor antagonists at the CP.
The choroid plexus (CP), located in the lateral, third, and fourth
ventricles, is the site of production of cerebrospinal fluid (CSF)
(Segal, 2000; Haselbach et al., 2001). It is well established that CP
acts as a barrier between the CSF and the circulating blood, and it is
referred to as the blood-CSF barrier (Suzuki et al., 1997; Ghersi-Egea
and Strazielle, 2001; Haselbach et al., 2001; Kusuhara and Sugiyama,
2001). The barrier function is achieved partly by the tight monolayer
of choroid plexus epithelial cells and partly by detoxification systems
consisting of metabolic enzymes and multispecific transporters (Suzuki et al., 1997; Ghersi-Egea and Strazielle, 2001; Haselbach et al.,
2001; Kusuhara and Sugiyama, 2001).
Histamine H2-receptor antagonists have been used clinically to cure
duodenal ulcers and gastric acid hypersecretion, and adverse effects
by H2-receptor antagonists on the central nervous system, ranging
from mild dizziness, restlessness, and mental confusion to advanced
symptoms such as myoclonic twitching and seizure, have been reported (Grimson, 1977; Schentag et al., 1979; McGuigan, 1981).
Schentag et al. (1979) reported that the concentration of cimetidine in
the CSF is related to the mental status. We demonstrated previously
that a saturable mechanism is involved in the elimination of cimetidine from the CSF after intracerebroventricular administration (Su-
zuki et al., 1985, 1988). Transport studies revealed that the uptake of
cimetidine by the isolated rat choroid plexus is saturable, and the
efflux transport across the CP has been considered to account for the
saturable elimination mechanism of cimetidine from the CSF (Suzuki
et al., 1986). Organic anions such as benzylpenicillin and p-aminohippurate (PAH) inhibit the uptake of cimetidine by the isolated rat
choroid plexus, whereas organic cations such as tetraethylammonium
(TEA) and N-methylnicotinamide have no such effect (Suzuki et al.,
1986). It is likely that organic anion transporter(s) play a major role in
regulating the CSF concentration of cimetidine at the CP.
We have isolated rat organic anion transporter 3 (rOat3; Slc22a8),
the third isoform of the Oat/OAT family, from the rat brain cDNA
library by homology cloning (Kusuhara et al., 1999). Functional
expression of rOat3 in Xenopus laevis oocytes and mammalian cells
has revealed its broad substrate specificity for organic anions, including PAH and benzylpenicillin (Kusuhara et al., 1999; Burckhardt and
Burckhardt, 2003; Dantzler and Wright, 2003). In the CP, although
mRNA expression of all the Oat isoforms (rOat1⬃rOat3) has been
detected (Sweet et al., 2002; Choudhuri et al., 2003), rOat3 is the most
abundant isoform (Choudhuri et al., 2003). rOat3 has been shown to
be expressed on the brush border membrane of the CP (Nagata et al.,
2002). Localization of rOat3 in the CP suggests its involvement in the
uptake process at the CP. Since the spectrum of inhibitors and kinetic
This work was supported by grant-in-aids from the Ministry of Health, Labor
and Welfare of Japan and a grant-in-aid for Scientific Research (B) (KAKENHI
15390035).
ABBREVIATIONS: CP, choroid plexus; CSF, cerebrospinal fluid; PAH, p-aminohippurate; TEA, tetraethylammonium; Oat, organic anion transporter; MEP, molecular electrostatic potential(s); LC-MS, liquid chromatography-mass spectometry; Oct, organic cation transporter.
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The choroid plexus (CP) acts as a site for the elimination of xenobiotic organic compounds from the cerebrospinal fluid (CSF). The
purpose of the present study is to investigate the role of rat organic
anion transporter 3 (rOat3; Slc22a8) in the uptake of H2-receptor
antagonists (cimetidine, ranitidine, and famotidine) by the isolated
rat CP. Saturable uptake of cimetidine and ranitidine was observed
in rOat3-LLC with Km values of 80 and 120 ␮M, respectively, whereas
famotidine was found to be a poor substrate. The steady-state concentration of the H2-receptor antagonists in the CSF was significantly
increased by simultaneously administered probenecid, although it did
not affect their brain and plasma concentrations. Saturable uptake of
cimetidine and ranitidine was observed in the isolated rat CP with Km
values of 93 and 170 ␮M, respectively, whereas 50% of the uptake of
CARRIER-MEDIATED UPTAKE OF H2-RECEPTOR ANTAGONISTS BY THE CP
Materials and Methods
Materials. [3H]Cimetidine (16.5 Ci/mmol) and [14C]urea (52 mCi/mmol)
were purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). All cell culture media and reagents were obtained from
Invitrogen (Carlsbad, CA), except fetal bovine serum (Cansera International
Inc., Ontario, Canada). All other chemicals and reagents were of analytical
grade and readily available from commercial sources.
Calculation of Molecular Electrostatic Potential (MEP) of H2-Receptor
Antagonists. The starting structures of H2-receptor antagonists were built up
on the basis of standard bond lengths and angles, and the structures of
H2-receptor antagonists were optimized using the AM1 Hamiltonian and
conductor-like screening model. Their molecular electrostatic potentials at pH
7.4 were calculated using the modified neglect of diatomic overlap Hamiltonian. All calculations were carried out by the MOPAC97 (CS Chem3D Pro;
CambridgeSoft Corporation, Cambridge, MA).
Uptake Studies by rOat3-LLC. rOat3-LLC was established previously,
and all the procedures have been described in detail (Sugiyama et al., 2001).
Cells were seeded on a 12-well dish (BD Biosciences, Franklin Lakes, NJ) at
a density of 1.2 ⫻ 105 cells/well and cultured for 3 days. Sodium butyrate (5
mM) was added to the culture medium to induce expression of the transporter
24 h before starting the experiments (Sugiyama et al., 2001). Uptake was
initiated by adding medium containing ligands, with or without inhibitors, after
the cells had been washed twice and preincubated with Krebs-Henseleit buffer
at 37°C for 15 min. This buffer consists of 142 mM NaCl, 23.8 mM Na2CO3,
4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES, 5 mM
glucose, and 1.53 mM CaCl2 adjusted to pH 7.4. The uptake was terminated at
a designated time by adding ice-cold Krebs-Henseleit buffer. For [3H]cimetidine uptake, cells were dissolved in 500 ␮l of 1 N NaOH, kept overnight,
neutralized with 250 ␮l of 2 N HCl, and then aliquots (500 ␮l) were transferred
to scintillation vials. The radioactivity associated with the cells and medium
was determined in a liquid scintillation spectrophotometer (LS6000SE; Beckman Coulter, Fullerton, CA) after adding scintillation fluid (Hionic-Fluor;
PerkinElmer Life and Analytical Sciences, Boston, MA) to the vials. For the
determination of the uptake of ranitidine and famotidine, cells were dissolved
in 200 ␮l of 0.2 N NaOH, and aliquots (50 ␮l) were used for LC-MS
quantification as described below. The remaining portions of cell lysate were
used to determine the protein concentration by the method of Lowry, with
bovine serum albumin as a standard. Ligand uptake was given as the cell-tomedium concentration ratio determined as the amount of ligand associated
with cells divided by the medium concentration.
Uptake Studies by Isolated Rat CP. Male Sprague-Dawley rats weighing
250 to 300g were purchased from SLC (Shizuoka, Japan). The CP was isolated
from the lateral ventricles and incubated at 37°C for 1 min in 500 ␮l of
artificial CSF, which consists of 122 mM NaCl, 25 mM NaHCO3, 10 mM
glucose, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4, and
10 mM HEPES (pH 7.3), equilibrated with 95% O2/5% CO2. Radiolabeled
ligands, with or without inhibitors, were added simultaneously to initiate
uptake. The uptake of [3H]cimetidine and [14C]TEA by isolated rat CP was
examined by centrifugal filtration as described previously (Nagata et al., 2002).
The tissue-to-medium concentration ratio of [3H]cimetidine and [14C]TEA was
calculated with [14C]urea or [3H]water as a cell water space marker and
corrected for the adherent water space. The 3H and 14C activity in the
specimens was determined in a liquid scintillation spectrophotometer. To
determine the uptake of nonradiolabeled H2-receptor antagonists, the rapid
filtration method was used since the silicon oil, used in the centrifugal filtration
method, disturbed the quantification by LC-MS. The uptake of cimetidine,
ranitidine, and famotidine by isolated rat CP was terminated by rapid filtration
using a vacuum manifold; then the CP was rinsed three times with 300 ␮l of
artificial CSF, the CP was dissolved in 100 ␮l of 0.2 N NaOH, and aliquots (50
␮l) were subjected to LC-MS quantification as described below. The remaining portions of lysate were used to determine the protein concentration by the
method of Lowry, with bovine serum albumin as a standard.
Constant Infusion Study in Rats. Rats were lightly anesthetized with
ether, and the left femoral vein was cannulated with polyethylene tubing
(PE-50; BD Biosciences). The priming dose of the H2 antagonist and probenecid was 2 and 15 mg/kg, respectively. The H2 antagonist and probenecid
were given to rats through the femoral vein cannula at 2 and 30 mg/h/kg,
respectively, for 3 h. The dose regimen was designed to obtain a CSF
concentration of probenecid sufficient to inhibit rOat3. Blood samples (300 ␮l)
were collected from the tail vein at 1 and 2 h during the infusion. At the end
of the experiment, blood samples were collected from the abdominal vein, and
animals were sacrificed by bleeding the abdominal aorta under ether anesthesia. Following sacrifice, 50- to 100-␮l aliquots of CSF were obtained by
cisternal puncture using insulin syringes (0.5-ml syringe with a 29-gauge ⫻
1/2-inch attached needle; TERUMO Corporation, Tokyo, Japan), and whole
brains were removed and weighed. To obtain plasma, blood was centrifuged at
10,000g for 5 min. For determination of the unbound plasma concentration,
aliquots (0.5 ml) of plasma specimens were subjected to filtration (2500 rpm,
10 min) (MPS-1; Millipore Corporation, Bedford, MA). The brain samples
were homogenized in three volumes of water using a Polytron homogenizer
(Brinkmann Instruments, Westbury, NY). The concentrations of H2-receptor
antagonists and probenecid in plasma, plasma ultrafiltrate, and brain homogenate were quantified by LC-MS as described below.
Quantification of H2-Receptor Antagonists and Probenecid by LC-MS.
The quantification of cimetidine, ranitidine, famotidine, and probenecid was
performed by a high-performance liquid chromatograph (Alliance 2690; Waters, Milford, MA) connected to a mass spectrometer (ZMD; Micromass UK
Ltd., Manchester, UK). Aliquots (50 ␮l) of samples containing H2-receptor
antagonists were precipitated by adding 100 ␮l of methanol containing an
internal standard (famotidine for cimetidine and ranitidine and cimetidine for
famotidine), mixed, and centrifuged, and 20 ␮l of the supernatants was
subjected to LC-MS. Aliquots (50 ␮l) of samples containing probenecid were
precipitated by adding 100 ␮l methanol, mixed, and centrifuged, and aliquots
(10 ␮l) of the supernatants were diluted with 990 ␮l of methanol, 20 ␮l of
which was subjected to LC-MS. High-performance liquid chromatography
analysis was performed on an Waters Xterra MS C18 column (2.5 ␮m, 3 mm
i.d., 30 mm) at room temperature. Elution was performed with a 0 to 100%
linear gradient of 10 mM ammonium acetate/methanol over 4 min at 0.8
ml/min. A portion of the eluent (split ratio ⫽ 1:3) was introduced into the MS
via an electrospray interface. Detection was performed by selective ion monitoring in positive ion mode (m/z: 253, 315, 338, and 286 for cimetidine,
ranitidine, famotidine, and probenecid, respectively).
Kinetic Analyses. Kinetic parameters were obtained using the MichaelisMenten equation v ⫽ Vmax ⫻ S/(Km ⫹ S) ⫹ Pdif ⫻ S, where v is the uptake rate
of the substrate (pmol/min/mg protein or pmol/min/␮l tissue), S is the substrate
concentration in the medium (␮M), Km is the Michaelis-Menten constant
(␮M), and Vmax is the maximum uptake rate (pmol/min/mg protein or pmol/
min/␮l tissue). Pdif represents the uptake clearance corresponding to the
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parameters for the uptake of PAH and benzylpenicillin by the isolated
rat CP were similar to those for rOat3, it has been hypothesized that
rOat3 accounts for their uptake by the isolated rat CP (Nagata et al.,
2002). Furthermore, the finding that the isolated choroid plexus from
mOat3 knockout mice was unable to cellularly accumulate fluorescein
supports the role of Oat3 in the CP (Sweet et al., 2002). H2-receptor
antagonists have been referred to as bisubstrates recognized by both
renal organic anion and cation transporters (Ullrich et al., 1993).
Indeed, cimetidine is a substrate of rOat3 as well as organic cation
transporter(s) (Grundemann et al., 1999; Kusuhara et al., 1999).
Therefore, it is likely that rOat3 plays a major role in the uptake of
cimetidine and other H2-receptor antagonists by the isolated rat CP.
The primary purpose of the present study was to investigate the
importance of rOat3 in regulating the concentration of H2-receptor
antagonists (cimetidine, ranitidine, and famotidine). We examined
whether ranitidine and famotidine are substrates of rOat3 in LLC-PK1
cells expressing rOat3 (rOat3-LLC). rOat3-LLC exhibits specific uptake of ranitidine and famotidine, but the transport activity of famotidine was quite low compared with that of cimetidine and ranitidine.
Steady-state concentrations of H2-receptor antagonists in the CSF and
plasma were determined in rats treated with or without probenecid, a
potent inhibitor of rOat3. The uptake of H2-receptor antagonists was
investigated using the isolated rat CP, and the kinetic parameters and
spectrum of inhibitors were compared with those for benzylpenicillin.
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NAGATA ET AL.
nonsaturable component (␮l/min/mg protein or ␮l/min/␮l tissue). To obtain
the kinetic parameters, the equation was fitted to the uptake velocity using a
MULTI program (Yamaoka et al., 1981). The input data were weighted as the
reciprocals of the observed values, and the Damping Gauss-Newton method
algorithm was used for fitting. Inhibition constants (Ki) were calculated by
assuming competitive inhibition using the equation CL⫹inh ⫽ CL/(1 ⫹ I/Ki) ⫹
Pdif, where CL represents the uptake clearance and ⫹inh represents the value in
the presence of inhibitor. I represents the concentration of inhibitor (␮M). The
substrate concentration was low compared with its Km value in the inhibition
study.
Results
Molecular Electrostatic Potential of H2-Receptor Antagonists.
Figure 1 illustrates the chemical structures of the H2-receptor antagonists and their isoelectric lines at 2.8 atomic units calculated at pH
7.4. There was a region showing negative MEP in the chemical
structures of H2-receptor antagonists (Fig. 1; indicated by blue in the
cyanoimine group for cimetidine, the nitro group for ranitidine, and
the imine and sulfonamide groups for famotidine).
Uptake of H2-Receptor Antagonists (Cimetidine, Ranitidine,
and Famotidine) by rOat3-LLC. Figure 2 shows the time profiles of
the uptake of cimetidine, ranitidine, and famotidine by vector- and
rOat3-LLC. The uptake of cimetidine, ranitidine, and famotidine was
significantly greater in rOat3-LLC than in vector-LLC, although the
absolute value of famotidine uptake was quite small compared with
that of cimetidine and ranitidine (Fig. 2). For further analyses, the
uptake of cimetidine and ranitidine was determined at the earliest
time, both technically and practically (3 min for cimetidine and 5 min
for ranitidine). The uptake of cimetidine and ranitidine was saturable
(Fig. 3), and kinetic analyses revealed that the Km and Vmax values of
cimetidine and ranitidine by rOat3-LLC were 79.2 ⫾ 17.8 and 121 ⫾
36 ␮M and 150 ⫾ 29 and 367 ⫾ 95 pmol/min/mg protein, respectively. The uptake clearance corresponding to the saturable component (Vmax/Km) for cimetidine and ranitidine was 1.89 ⫾ 0.30 and
3.03 ⫾ 0.39 ␮l/min/mg protein, respectively, whereas that corre-
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FIG. 1. Molecular structures and molecular electrostatic potential of H2-receptor
antagonists. MEP of cimetidine, ranitidine, and famotidine at pH 7.4 were calculated
using the MNDO Hamiltonian. The figure illustrates the isoelectric line at 2.8
atomic units. The colors indicate the charge of their electrostatic potential: red is
positive and blue is negative.
sponding to the nonsaturable component was 1.07 ⫾ 0.03 and
0.381 ⫾ 0.023 ␮l/min/mg protein, respectively.
Effect of Organic Anions and Cations on the Uptake of Cimetidine by rOat3. Benzylpenicillin and ranitidine inhibited the rOat3mediated [3H]cimetidine uptake in a concentration-dependent manner
(Fig. 4, a and b), whereas TEA had no inhibitory effect at the
concentrations examined (0.1 to 10 mM; data not shown). The Ki
values of benzylpenicillin and ranitidine for cimetidine uptake by
rOat3-LLC were determined to be 76.7 ⫾ 13.2 and 119 ⫾ 44 ␮M,
respectively. Famotidine only weakly inhibited the uptake of cimetidine by rOat3-LLC (Fig. 4c).
Effect of Probenecid on the Plasma, Brain, and CSF Concentrations of H2-Receptor Antagonists. Rats were given H2-receptor
antagonists by constant infusion with or without probenecid. The
plasma concentrations of H2-receptor antagonists reached steady-state
within 3 h (data not shown), whereas the plasma concentration of
probenecid showed a gradual increase during infusion (355 ⫾ 107
␮M at 1 h, 514 ⫾ 143 ␮M at 2 h, and 651 ⫾ 246 ␮M at 3h). The
unbound plasma concentration and CSF concentration of probenecid
at 3 h were 154 ⫾ 26 and 57.3 ⫾ 2.0 ␮M, respectively. Probenecid
treatment did not affect the brain and plasma concentrations of the
H2-receptor antagonists (Table 1), whereas their CSF concentrations
significantly increased (Table 1). The CSF-to-unbound plasma concentration ratio (CCSF/Cp,fu) significantly increased by 3.7-fold for
cimetidine, 4.3-fold for ranitidine, and 2.5-fold for famotidine (Table 1).
Uptake of H2-Receptor Antagonists by the Isolated Rat CP. The
time profiles of the uptake of [3H]cimetidine and [14C]TEA by isolated rat CP are shown in Fig. 5a (uptake units: ␮l/␮l CP volume) and
those of cimetidine, ranitidine, and famotidine are shown in Fig. 5b
(uptake units: ␮l/mg protein). All the H2-receptor antagonists showed
time-dependent accumulation by the isolated rat CP, and their transport activities were in the following order: ranitidine ⬇ cimetidine ⬎
famotidine (Fig. 5b). The uptake of TEA by the isolated rat CP was
small compared with that of H2-receptor antagonists (Fig. 5a). The
uptake of [3H]cimetidine and ranitidine was saturable, and kinetic
analyses revealed that the uptake of [3H]cimetidine and ranitidine
consists of one saturable and one nonsaturable component (Fig. 6).
The Km and Vmax values and uptake clearance corresponding to the
nonsaturable component of [3H]cimetidine were 92.7 ⫾ 46.1 ␮M,
137 ⫾ 71 pmol/min/␮l tissue, and 0.581 ⫾ 0.123 ␮l/min/␮l tissue,
respectively, whereas the corresponding values for ranitidine were
171 ⫾ 57 ␮M, 1250 ⫾ 360 pmol/min/mg protein, and 1.49 ⫾ 0.26
␮l/min/mg protein. The uptake of famotidine was saturated at high
substrate concentrations (Table 2), but 50% of the total uptake remained at the highest concentration examined (1 mM, Table 2).
Effect of Organic Anions and TEA on the Uptake of Cimetidine
by Isolated Rat CP. The effect of benzylpenicillin and TEA was
examined with regard to the uptake of cimetidine, ranitidine, and
famotidine by the isolated rat CP (Table 2). Benzylpenicillin inhibited
the uptake of the H2-receptor antagonists in a concentration-dependent manner. TEA did not affect the uptake of H2-receptor antagonists. Furthermore, the effect of estradiol-17␤-glucuronide, estrone
sulfate, benzylpenicillin, PAH, ranitidine, and famotidine was also
examined with regard to the uptake of [3H]cimetidine by isolated rat
CP (Fig. 7). All organic anions, ranitidine, and famotidine showed a
concentration-dependent inhibition of the uptake of [3H]cimetidine by
the isolated rat CP (Fig. 7), and their inhibition constants are summarized in Table 3. The Ki and Km values for the uptake of benzylpenicillin and cimetidine by the isolated rat CP were comparable (Table
3).
CARRIER-MEDIATED UPTAKE OF H2-RECEPTOR ANTAGONISTS BY THE CP
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FIG. 3. Concentration dependence of the uptake of cimetidine and ranitidine by rOat3-LLC. The cellular accumulation of [3H]cimetidine (a) and ranitidine (b) by
rOat3-LLC for 3 and 5 min was determined at different substrate concentrations. The concentration dependence of the uptake of cimetidine and ranitidine by rOat3-LLC
is shown as Eadie-Hofstee plots in the inset. Kinetic analyses revealed that the uptake of [3H]cimetidine and ranitidine consists of one saturable and one nonsaturable
component and follows the Michaelis-Menten equation. The solid, dotted, and broken lines represent the fitted line, the clearance of the nonsaturable component, and the
clearance of the saturable component obtained by nonlinear regression analysis, respectively. Each point represents the mean ⫾ S.E. (n ⫽ 3– 6).
FIG. 4. Effect of organic anions and cations on the uptake of cimetidine by rOat3. The cellular accumulation of [3H]cimetidine (1 ␮M) by rOat3 for 3 min was determined
in the presence and absence of nonradiolabeled compounds at the concentrations indicated. Symbols represent benzylpenicillin (a; 䡺), ranitidine (b; f), and famotidine
(c; Œ). The inhibition constants (Ki) of these compounds were calculated by assuming competitive inhibition. The solid lines represent the fitted line obtained by nonlinear
regression analysis. The details of the fitting are described under Materials and Methods. Each point represents the mean ⫾ S.E. (n ⫽ 3).
Discussion
In the present study, we reported that rOat3 is involved in the
uptake of H2-receptor antagonists (cimetidine, ranitidine, and famo-
tidine) by the isolated rat CP, and that drug-drug interaction causes an
increase in the CSF concentration of H2-receptor antagonists without
affecting their plasma concentration. rOat3 has been characterized by
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FIG. 2. Uptake of cimetidine, ranitidine, and famotidine by rOat3-LLC. The uptake of [3H]cimetidine (a; circle), ranitidine (b; square) and famotidine (c; triangle) by
rOat3-LLC (closed symbol) and vector-LLC (open symbol) was determined. The uptake was initiated by adding ligand ([3H]cimetidine (1 ␮M), ranitidine (10 ␮M), and
famotidine (10 ␮M) and terminated at designated times by adding ice-cold buffer. Each point represents the mean ⫾ S.E. (n ⫽ 3– 6). ⴱⴱ, p ⬍ 0.01 and ⴱⴱⴱ, p ⬍ 0.001,
significantly different from vector-LLC (unpaired t test).
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NAGATA ET AL.
TABLE 1
Total plasma, unbound plasma, CSF, and brain concentrations of H2-receptor antagonists after intravenous infusion in rats treated with or without probenecid
H2-receptor antagonists (2 mg/kg) were administered to rats by bolus intravenous administration followed by constant infusion (2 mg/h/kg) during 3 h. Probenecid (15 mg/kg) was
administered to rats by bolus intravenous administration followed by constant infusion (30 mg/h/kg) during 3 h, and the unbound plasma concentrations and CSF concentrations of probenecid
at 3 h were 154 ⫾ 26 and 57.3 ⫾ 2.0 ␮M, respectively. Each value represents the mean ⫾ S.D. (n ⫽ 3).
Cimetidine
Cp (␮M)
Cp,fu (␮M)
CCSF (nM)
Cbrain (nM)
CCSF/Cp,fu ratio
Ranitidine
Famotidine
Control
⫹Probenecid
Control
⫹Probenecid
Control
⫹Probenecid
3.71 ⫾ 1.20
2.25 ⫾ 0.60
39.1 ⫾ 19.8
96.5 ⫾ 27.1
0.0166 ⫾ 0.005
2.97 ⫾ 0.15
1.89 ⫾ 0.11
116 ⫾ 17**
99.9 ⫾ 12.5
0.0615 ⫾ 0.0121**
2.65 ⫾ 1.25
1.72 ⫾ 0.49
25.9 ⫾ 12.0
85.7 ⫾ 33.8
0.0147 ⫾ 0.0046
2.11 ⫾ 0.18
1.52 ⫾ 0.06
97.1 ⫾ 5.2***
83.9 ⫾ 10.9
0.0639 ⫾ 0.0046***
3.47 ⫾ 1.22
2.01 ⫾ 0.72
23.1 ⫾ 9.2
106 ⫾ 30
0.0114 ⫾ 0.0027
2.79 ⫾ 0.03
1.54 ⫾ 0.18
43.9 ⫾ 4.1*
102 ⫾ 18
0.0289 ⫾ 0.0057**
Cp, total plasma concentration; Cp,fu, unbound plasma concentration; CCSF, CSF concentration; Cbrain, brain concentration.
* p ⬍ 0.05, ** p ⬍ 0.01, and *** p ⬍ 0.001, significantly different from control rats (unpaired t test).
FIG. 6. Concentration dependence of cimetidine and ranitidine accumulation by the isolated rat CP. The accumulation of [3H]cimetidine (a) and ranitidine (b) in the isolated
rat CP for 3 and 5 min, respectively, was determined at various substrate concentrations. The concentration dependence for the uptake of [3H]cimetidine and ranitidine is
shown as Eadie-Hofstee plots in the inset. Kinetic analyses revealed that the uptake of [3H]cimetidine and ranitidine consists of one saturable and one nonsaturable
component and follows the Michaelis-Menten equation. The solid, dotted, and broken lines represent the fitted line, the clearance of the nonsaturable component, and the
clearance of the saturable component obtained by nonlinear regression analysis, respectively. Each point represents the mean ⫾ S.E. (n ⫽ 3).
its broad substrate specificity for organic anions, from amphipathic to
hydrophilic organic anions, and also a weakly basic compound, cimetidine (Kusuhara et al., 1999). In addition to cimetidine, ranitidine
was found to be a good substrate of rOat3, whereas famotidine seems
to be a poor substrate of rOat3 (Fig. 2). Because it is one of the clues
to understanding the interaction of such weak base or cationic compounds with rOat3, the MEP of the H2-receptor antagonists was
calculated (Fig. 1). The H2-receptor antagonists contain a region of
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
FIG. 5. Uptake of cimetidine ranitidine, famotidine, and TEA by the isolated rat CP. The rat CP was isolated from the lateral ventricles. a, uptake of [3H]cimetidine (F)
and [14C]TEA (⽧) by the isolated rat CP was determined by the centrifugal filtration method as described under Materials and Methods. The tissue-to-medium
concentration ratio of [3H]cimetidine and [14C]TEA was calculated with [14C]urea and [3H]water as cell water space markers, respectively, and corrected for the adherent
water space. b, uptake of cimetidine (10 ␮M; F), ranitidine (10 ␮M; f), and famotidine (10 ␮M; Œ) by the isolated CP was determined by the rapid filtration method
followed by quantification using LC-MS. Each point represents the mean ⫾ S.E. (n ⫽ 3).
CARRIER-MEDIATED UPTAKE OF H2-RECEPTOR ANTAGONISTS BY THE CP
TABLE 2
Effect of benzylpenicillin and TEA on the uptake of H2-receptor antagonists by
the isolated rat CP
The uptake of cimetidine, ranitidine, and famotidine by isolated rat CP was determined in
the presence and absence of benzylpenicillin and TEA at the concentrations indicated. The
substrate concentration of cimetidine, ranitidine, and famotidine was 10 ␮M, and the excess
amount of unlabeled substrates represents concentrations of 3, 3, and 1 mM for cimetidine,
ranitidine, and famotidine, respectively. Each value represents the mean ⫾ S.E. (n ⫽ 3).
Uptake (Percentage of Control)
Concentration
Cimetidine
Ranitidine
Famotidine
100 ⫾ 7
30.4 ⫾ 1.1
72.5 ⫾ 5.1
46.3 ⫾ 1.1
30.4 ⫾ 1.1
99.5 ⫾ 8.3
87.5 ⫾ 6.1
93.7 ⫾ 10.5
100 ⫾ 12
24.8 ⫾ 1.1
76.7 ⫾ 19
34.9 ⫾ 3.9
31.8 ⫾ 3.5
94.8 ⫾ 14
97.3 ⫾ 8.6
88.8 ⫾ 13.6
100 ⫾ 22
50.5 ⫾ 3.6
83.8 ⫾ 8.3
78.6 ⫾ 6.6
51.6 ⫾ 1.9
120 ⫾ 3
110 ⫾ 14
93.4 ⫾ 3.6
mM
Control
Excess cold
Benzylpenicillin
TEA
0.1
1
3
0.1
1
3
Probenecid has previously been reported to inhibit the apical-tobasal side of azidodeoxythymidine (corresponding to the efflux transport from the CSF side to the blood side under physiological conditions), resulting in an increase in the basal-to-apical transport
(Strazielle et al., 2003). Simultaneous administration of probenecid
caused a significant increase in the CSF concentration of all the
H2-receptor antagonists examined (Table 1). Since it did not affect the
brain and unbound plasma concentrations, it is likely that the effect of
probenecid is due to the inhibition of the efflux transport of H2receptor antagonists across the CP (Table 1). Strazielle et al. (2003)
also found that benzbromarone had an inhibitory effect for the apicalto-basal transport of azidodeoxythymidine. It is possible that benzbromarone treatment also causes an increase in the CSF concentration of
H2-receptor antagonists. Since the brain-to-unbound plasma concentration ratio of the H2-receptor antagonists was well below unity
(Table 1), it is possible that the efflux transport across the brain
capillaries limits their brain distribution; however, probenecid had no
effect on the brain concentration. Further studies are necessary to
investigate whether the H2-receptor antagonists undergo the efflux
across the brain capillaries and whether organic anion transporters,
including rOat3, contribute to this efflux.
The uptake, an initial process for elimination from the CSF, was
investigated using the isolated rat CP. Time-dependent uptake of the
H2-receptor antagonists was detected in the isolated rat CP (Fig. 5).
The saturable component accounts for a large part of the total uptake
of cimetidine and ranitidine (Fig. 6). The uptake of cimetidine by the
isolated rat CP was markedly inhibited by benzylpenicillin with a Ki
value similar to its own Km value for uptake by the isolated rat CP
(Nagata et al., 2002). Conversely, the Km value of cimetidine for the
uptake by the isolated rat CP was similar to its Ki value for the uptake
FIG. 7. Inhibitory effect of organic anions and cations on the uptake of cimetidine by the isolated rat CP. The accumulation of [3H]cimetidine (1 ␮M) by the isolated rat
CP for 3 min was determined in the presence and absence of nonradiolabeled compounds at the concentrations indicated. Symbols represent benzylpenicillin (a; 䡺), PAH
(b; 〫), estradiol-17␤-glucuronide (c; E), estrone sulfate (d; ‚), ranitidine (e; f), and famotidine (f; Œ). The solid lines represent the fitted line obtained by nonlinear
regression analysis. The details of the fitting are described under Materials and Methods. Each point represents the mean ⫾ S.E. (n ⫽ 3).
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
negative MEP in their chemical structures (Fig. 1). As Ullrich et al.
(1993) suggested, this site might play a key role for the substrate
recognition of H2-receptor antagonists by rOat3. Suzuki et al. (1987)
clearly demonstrated a linear correlation between the lipophilicity and
reciprocal number of Ki values of ␤-lactam antibiotics for the uptake
of benzylpenicillin by the isolated rat CP. Lipophilicity is likely to be
an important factor for recognition by rOat3 in the case of ␤-lactam
antibiotics. In contrast, the kinetic parameters of cimetidine and
ranitidine for their uptake by rOat3 were comparable, although they
had different cLogD values. Lipophilicity may not be a determinant
factor in the case of the H2-receptor antagonists.
1045
1046
NAGATA ET AL.
TABLE 3
Ki and Km values for the uptake of cimetidine, ranitidine, and benzylpenicillin by
the isolated rat CP
The effect of estrone sulfate, cimetidine, estradiol-17␤-glucuronide (E217␤G), PAH,
benzylpenicillin, and ranitidine was examined with regard to uptake by the rat isolated CP.
The Ki and Km values were determined by nonlinear regression analysis as described under
Materials and Methods. Data are taken from Fig. 7. Each value represents the mean ⫾ S.E.
(n ⫽ 3– 6).
Ki and Km values (␮M)
Estrone sulfate
Cimetidine
E217␤G
PAH
Benzylpenicillin
Ranitidine
a
b
Benzylpenicillina
Cimetidine
Ranitidine
22.3
44.4
33.0
406
111b
19.5 ⫾ 8.8
92.7 ⫾ 46.1b
36.3 ⫾ 15.7
281 ⫾ 74
140 ⫾ 97
49.6 ⫾ 15.1
171 ⫾ 57b
Parameters cited from Nagata et al. (2002).
Km value.
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Furthermore, the inhibition constants of the compounds listed in Table
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Reverse transcription-polymerase chain reaction analyses detected
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2001). The expression level of rOct2 in the CP was considerably
lower than that in the kidney, whereas that of rOct3 was the same for
all the tissues examined, although the absolute value was low
(Choudhuri et al., 2003). The low expression of Oct mRNA may
account for the lower uptake of TEA by the isolated rat CP compared
with that of the H2-receptor antagonists (Fig. 5). Although cimetidine
is a substrate of rOct2 (Grundemann et al., 1999), the effect of TEA
on the uptake of the H2-receptor antagonists by the isolated rat CP was
minimal, even at a concentration sufficient to saturate TEA uptake by
primary cultured choroid epithelial cells (Table 2) (Villalobos et al.,
1997). Therefore, the contribution of rOcts to the total uptake of
H2-receptor antagonists by the isolated rat CP is minimal, although
they are involved.
rOat3 has been shown to be expressed on the basolateral membrane
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(Hasegawa et al., 2002, 2003). However, probenecid treatment did not
affect the steady-state plasma concentration of H2-receptor antagonists, although the unbound plasma concentration was sufficient to
inhibit rOat3-mediated uptake (Table 1). This result is in a good
agreement with the previous report by Boom and Russel (1993). They
examined the uptake of cimetidine by freshly isolated rat proximal
tubular cells and demonstrated that the major fraction of cimetidine
uptake (approximately 50%) was inhibited by TEA (Boom and Russel, 1993). Probenecid was only a weak inhibitor, with an IC50 value
(700 ␮M) greater than the unbound plasma concentration employed in
this study. Organic cation transporter(s) will play a major role in the
renal uptake of cimetidine, whereas the uptake by the isolated rat CP
is totally accounted for by the organic anion transporter. This unique
phenomenon is entirely due to the unique nature of the bisubstrate,
which is a substrate of both organic anion and cation transporters.
The results of the present study suggest the possibility of drug-drug
interactions between the H2-receptor antagonists and organic anions
that cause an increase in the CSF concentration of H2-receptor antagonists without affecting their plasma concentration profiles, as in the
case of probenecid. The risk of mental confusion with cimetidine in
patients with renal or hepatic dysfunction is higher than that in normal
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Indeed, some organic anions (e.g., quinolinic acid) are known to be
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In conclusion, cimetidine and ranitidine are good substrates for
rOat3, whereas famotidine is a poor substrate for this transporter. The
efflux transport across the CP plays an important role in regulating the
CSF concentration of H2-receptor antagonists. rOat3 is the most likely
candidate transporter for the uptake of H2-receptor antagonists by the
isolated rat CP.
CARRIER-MEDIATED UPTAKE OF H2-RECEPTOR ANTAGONISTS BY THE CP
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